CN112673247A - Systems, devices, and methods for off-axis illumination in flow cytometry - Google Patents

Abstract

A flow cytometer comprising a laser beam impinging a sample stream at an angle of 15 ° -70 °, optionally 30 °, from an orthogonal plane where light intersects the fluid.

Description

Systems, devices, and methods for off-axis illumination in flow cytometry

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 62/695,937 entitled "SYSTEM, APPARATUS AND METHOD FOR OFF AXIS ILLUMINATION IN FLOW circulation," filed on 7/10 of 2018, the entire contents of which are incorporated herein by reference.

Technical Field

The disclosed technology relates to flow cytometry. More particularly, in one embodiment, the disclosed technology relates to off-axis illumination and capture of scattered and fluorescent light in flow cytometry.

Background

Flow cytometry measures and analyzes a variety of physical properties of individual particles (typically cells) as they flow through a light beam in a fluid stream. The measured characteristics include the relative size, relative particle size or internal complexity of the particles, and relative fluorescence intensity. The stream of particles is expanded so that the particles pass through the beam one after the other in rapid succession. In one example, 100 and 10,000 particles per second traveling at a velocity of 1-10m/s are contained in a 100 μm flow with a 10 μm core. Light induces a small optical signal each time a particle passes through the beam. One or more detectors are positioned such that they view the point where the laser path and the cell trajectory intersect. The detector may register scattered and fluorescence signals of different colors and/or different polarizations. The signal provides an indication of the number and type of cells present in the sample.

Flow cytometry is commonly used to count and analyze blood cells. This technique plays a key role in HIV diagnosis and classification of blood cancers. It is also an important research tool. In recent years, flow cytometry has been successfully used to analyze plankton in seawater and freshwater samples.

In some prior art arrangements such as that shown in fig. 1, theilluminating light 14 is orthogonal to the trajectory of theparticles 20. In these instruments, the detector(s) 22 are also orthogonal to the particle path. One such arrangement is disclosed in U.S. patent No. 9,453,789, which is incorporated herein by reference. When illuminating the flow of a fluidic cytometer along an orthogonal axis, it is necessary to mask the detector 22 with amasking strip 24 to eliminate the light from the light beam and its scattering/reflection. This narrows or limits the useful aperture 26 of the detector 22. Scattering cannot be completely eliminated, resulting in background signal noise.

In some other prior art arrangements such as that shown in fig. 2, the detectors 122a, 122b detect light emitted along the direction of travel. According to Muriella et al (such as U.S. patent No. 5,475,487, which is incorporated herein by reference) and van den Engh et al (such as U.S. publication No. 2014/0320861, which is incorporated herein by reference), detectors 122a, 122b may be positioned along the axis of the liquid jet (such as above and below the laser/jet intersection location) in order to preserve the entire aperture 126a, 126b of detectors 122a, 122 b. In this case, the collected light reaches the detector by internal reflection inside the liquid jet. This method is rarely used.

The above arrangement of fig. 1 and 2 has been the standard for most technologies that exist for about fifty (50) years. The orthogonal arrangement facilitates optical design. The mechanisms that move and align the optical components (e.g., lens that focuses the beam(s), mechanism that focuses the detector onto the field of measurement, and other adjustment mechanisms) are easiest to design, manufacture, and adjust when all components move along orthogonal paths.

Disclosure of Invention

Despite the many advantages of conventional flow cytometry, there is at least one major drawback to orthogonal optical geometries. In the prior art, the laser light is scattered by the sample flow. Since the two are orthogonal, the scattering lies in a plane around the fluid flow. In the prior art, the same plane also has detectors and other optical paths. If no measurement is made, the scattered light will enter the detector. The detector will be masked since the scatter is significantly brighter than the signal generated by the particles. Therefore, all instruments with orthogonal optical geometries require scattering bars that mask scattered light and filters that block unwanted colors. Designing and adjusting the scattering bars or masks is a delicate technique and greatly affects the quality of the scattering signal. This is particularly problematic when multiple laser beams are used. Poor adjustment of the scatter mask results in very different data for numerous laboratories. In addition, scattered light from the sample flow is directed to the surroundings and may escape from the measurement region, thereby exposing the technician to the scattered laser light.

For these reasons, conventional flow cytometry requires improvements to overcome the above and other deficiencies of the prior art. The disclosed technology addresses and corrects the above and other problems of the prior art.

In one embodiment, the disclosed technology relates to a flow cytometer comprising a light source configured to emit at least one light beam along a first axis. The flow cytometer may also include a channel configured to allow a particle-laden liquid to pass therethrough. The channel defines or extends at least along a second axis. The second axis may extend at an angle greater than 0 ° and less than 90 ° relative to the first axis. The flow cytometer may also include at least one detector configured to observe at least a point at which the light beam and the liquid intersect.

In another embodiment, the disclosed technology relates to a flow cytometer including a light source configured to emit at least one light beam along a first axis. The flow cytometer may also include a channel configured to allow a particle-laden liquid to pass therethrough. The channel defines or at least extends along a second axis. The flow cytometer may also include at least one detector configured to observe at least a point at which the light beam and the liquid intersect. Optionally, no masking or scattering bars are used, such that the entire aperture of the at least one detector exposes at least one beam.

Drawings

The foregoing summary, as well as the following detailed description of the disclosed technology, will be better understood when read in conjunction with the appended drawings, wherein like numerals denote like elements throughout. For the purpose of illustrating the disclosed technology, there are shown in the drawings various exemplary illustrative embodiments. It should be understood, however, that the disclosed technology is not limited to the precise arrangements and instrumentalities shown. In the drawings:

FIG. 1 is a schematic diagram of an arrangement in the prior art;

FIG. 2 is a schematic diagram of another arrangement in the prior art;

FIG. 3 is a schematic diagram of an arrangement in accordance with one embodiment of the disclosed technology;

FIG. 4 is a schematic diagram of an arrangement according to another embodiment of the disclosed technology; and

FIG. 5 is an image of at least a portion of a system or device in accordance with embodiments of the disclosed technology.

Detailed Description

Although the systems, devices, and methods are described herein by way of example and embodiments, those skilled in the art will recognize that the disclosed technology is not limited to the embodiments or figures described. On the contrary, the disclosed technology is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the appended claims. Features of any one embodiment disclosed herein may be omitted or combined with another embodiment.

Any headings used herein are for organizational purposes only and are not meant to limit the scope of the description or the claims. As used herein, the word "may" is used in a loose sense (i.e., meaning possible) rather than in a mandatory sense (i.e., meaning necessary). Unless specifically set forth herein, the terms "a," an, "and" the "are not limited to one element but are to be construed to mean" at least one. The terminology includes the words above mentioned, derivatives thereof, and words of similar import.

In one embodiment, the disclosed technology relates to an instrument in which a laser beam bombards a sample stream at an angle. For example, the angle may optionally be a first angle α (such as about or exactly 30 °) measured from the orthogonal plane P, or from the flow axis a₂A second angle theta measured (such as about or exactly 60 deg.). Alternatively, the second angle θ may be in the range of 30 ° -75 °, alternatively 37 ° or 34 °, relative to the flow axis. Alternatively, the first angle α may be in a complementary range or a complementary value.

In some cases, a second angle θ of 60 ° is optimal but not necessary as it provides most of the advantages described herein and is relatively easy to construct. From a physical point of view, a second angle θ of 34 ° is optimal but not necessary, since there is perfect optical coupling of the laser and the jet, and all reflections can be eliminated by adjusting the polarization direction of the incident light to be in the plane of the jet surface. A second angle theta of 34 deg. is optimal but not necessary if the sample flow is contained in the glass channel. If the sample stream is a liquid in an air jet, a second angle θ of 37 ° is optimal but not necessary.

Alternatively, the system may utilize a cylindrical beam dump to capture the direct laser light as well as the light scattered by the stream. In one embodiment, multiple detectors may be placed in, for example, a ring around the point of illumination due to the increased spacing created by placing the laser optics above the orthogonal intersection planes. Such a system can be easily tuned and optimized and does not require scattering bars, thereby greatly increasing the effective numerical aperture of the detector. In addition, the disclosed techniques provide excellent signal-to-noise ratio measurements on small, dim particles that are difficult to measure on prior art flow cytometers due to the low background scattered light generated or induced. Alternatively, two opposing detectors may face each other along a common axis (e.g., see fig. 4).

Off-axis illumination of the disclosed technology has many advantages. For example, first, the scatter from the flow is directed downward in a conical shape and can be collected in a cylindrical light trap. The risk of light escaping and obscuring the operator is minimized. Second, there is more room for a detector since the optics that bring the light to the measurement point are out of the orthogonal plane. In conventional instruments, the plane is crowded with components and only two detector channels may be present. When the illumination optics are above the detection plane, 4 or even 5 detectors (which include backscatter detectors that are not possible in orthogonal geometries) become possible. Third, the angle of incidence of the laser beam is closer to the critical angle (where there is no scattering from the liquid surface). The non-orthogonal arrangement has less surface scattering than the orthogonal arrangement. Fourth, there is no scattered light in the plane of the detector, as it is contained in a downward cone and can be captured in a cylindrical beam dump. In many cases, no scattering mask or laser blocking filter is required. Therefore, the problem of selecting an appropriate color band (chromatographic band) for signal detection becomes easier. In addition, the use of multiple detectors enables different detection colors to be distributed over the multiple detector assemblies. In some prior art systems, complex filter sets must be designed to use a single detector channel for multiple colors, but the non-orthogonal design of one embodiment of the disclosed technology may use multiple detectors, each specialized for use in a limited range of colors.

Referring now in detail to the drawings in which like reference numerals refer to like parts throughout, FIG. 3 illustrates one embodiment of at least a portion of a flow cytometer, generally designated 210, of the disclosed technology. Theflow cytometer 210 may include a first axis a configured to be along₁A light source 212 (such as, but not limited to, a laser) emitting at least one light beam 214 (e.g., a laser beam) and a channel 216 configured to allow a fluid 218 (such as a liquid or a gas) carrying one or more particles 220 to pass therethrough. The passage 216 defines a flow axis or second axis A₂Or the passage being along the flow axis or the second axis A₂And (4) extending. The fluid 218 may be a jet or may be contained within, for example, a glass capillary that forms the channel 216. Theflow cytometer 210 may include: at least one detector 222 having an entire aperture or a fully exposed aperture 226; taper 228 (optionally about or exactly 60 degrees); and abeam dump 230. Alternatively, thebeam dump 230 may be cylindrical.

First axis A₁Relative to the second axis A₂The second angle θ of extension is greater than zero and less than 90 °. Optionally, the second angle θ is between 20 ° -75 °, optionally between 34 ° -70 °, and optionally 30 °. At least one focusing lens 232 may be positioned between the light source 212 and the channel 216 (e.g., spaced apart from the light source 212 and the channel 216) at the first axis a₁In (1). Of course, the second angle θ may be calculated as 90 ° minus the first angle α.

When the excitation laser beam is elevated or positioned above or away from the orthogonal plane P of the laser/jet intersection at the first angle a or the second angle θ, the exit beam and the scattered beam are contained within the cone 228 and may be intersected by thebeam dump 230. The detector 222 can now utilize its entire aperture 226. Scattered light and background noise are completely eliminated. No masking or scattering bars are required.

Fig. 4 illustrates another embodiment of the disclosed technology. In fig. 4, similar or identical structures between the embodiment of fig. 3 and the embodiment of fig. 4 are distinguished by reference numerals that are one hundred (100) greater than those of fig. 3. The description of some of the similarities between the embodiment of fig. 3 and the embodiment of fig. 4 may be omitted here for convenience and brevity only.

As shown in fig. 4, when the excitation laser beam is sufficiently elevated above an orthogonal plane passing through the laser/jet intersection location, the plane is free of optical components, and additional detectors 322a, 322b may be placed around the intersection point. The use of multiple detectors allows signals to be collected at different angles and simplifies the design of the filter scheme for a particular color band. This arrangement also allows backscatter detection. In the current embodiment, up to six detectors may be placed at selectable angles around the intersection. Each of these detectors may have an entire aperture 326a, 326b exposed to thebeam 314 or available for thebeam 314. Prior art instruments have at most only two quadrature detectors.

Fig. 5 shows an image of another embodiment of the disclosed technology. Similar or identical structures between the embodiment of fig. 4 and the embodiment of fig. 5 are distinguished in fig. 5 by reference numerals that are one hundred (100) greater than the reference numerals of fig. 4. For convenience and brevity purposes only and not by way of limitation, descriptions of certain points of similarity between the embodiment of fig. 4 and the embodiment of fig. 5 are omitted herein.

Fig. 5 shows fluid 418 emanating from a nozzle orpassage 416.

The following exemplary embodiments further describe optional aspects of the disclosed technology and are part of this detailed description. Although these exemplary embodiments are not technically claim the present application, these exemplary embodiments are set forth in a format that is substantially similar to the claims (each with a numeric label followed by the letter a). The following exemplary embodiments are referred to in a dependent relationship with each other as "embodiments" and not as "claims".

A flow cytometer, comprising:

an optical path extending along a first axis; and

a fluid flow path extending along a second axis,

wherein the first axis extends at an angle greater than 0 ° and less than 90 ° relative to the second axis.

The flow cytometer according to example 1A, further comprising:

at least one detector configured to observe at least a point where the light beam and the liquid intersect.

The flow cytometer according to example 1A or 2A, wherein the angle is between 20 ° -75 °.

A method of eliminating the need to partially shield a detector of a flow cytometry system, the method comprising:

the light beam is arranged to extend at an angle of more than 0 ° and less than 90 ° relative to the fluid flow path in which the particles are carried.

The method of embodiment 1B, wherein the angle is between 20 ° -75 °.

The method of embodiment 2B, wherein the angle is between 34 ° -70 °.

The method of embodiment 3B, wherein the angle is about 60 °.

The method of embodiment 3B, wherein the angle is about 37 °.

A system configured to eliminate the need for partially shielding a detector of a flow cytometer, the system comprising:

an optical path extending along a first axis; and

a fluid flow path extending along a second axis,

wherein the first axis extends at an angle greater than 0 ° and less than 90 ° relative to the second axis.

The system of embodiment 1C, wherein the angle is between 20 ° -75 °.

The system of embodiment 2C, wherein the angle is between 34 ° -70 °.

The system of embodiment 3C, wherein the angle is about 60 °.

The system of embodiment 3C, wherein the angle is about 37 °.

The system of any of embodiments 1C to 5C, further comprising:

at least one detector configured to observe at least a point at which the light path and the fluid flow path intersect.

While the disclosed technology has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is understood, therefore, that the disclosed technology is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the disclosed technology as defined by the appended claims.

Claims (17)

1. A flow cytometer, comprising:

a light source configured to emit at least one light beam along a first axis;

a channel configured to allow passage of a particle-laden liquid therethrough, the channel extending along a second axis extending at an angle greater than 0 ° and less than 90 ° relative to the first axis; and

at least one detector configured to observe at least a point at which the light beam and the liquid intersect.

2. The flow cytometer of claim 1 wherein the at least one detector comprises:

at least two spaced apart detectors.

3. The flow cytometer of any of the preceding claims, wherein the angle is between 20 ° -75 °.

4. The flow cytometer of claim 3 wherein the angle is between 34 ° -70 °.

5. The flow cytometer of claim 4 wherein the angle is about 60 °.

6. The flow cytometer of claim 4 wherein the angle is about 37 °.

7. The flow cytometer of claim 1 wherein the light source is at least one laser.

8. The flow cytometer of claim 1 wherein no masking or scattering bars are used.

9. The flow cytometer of claim 1 wherein the entire aperture of the at least one detector is exposed to the at least one beam.

10. The flow cytometer of claim 1 wherein the at least one detector comprises six detectors.

11. The flow cytometer of claim 1, further comprising at least one focusing lens positioned in the first axis between the light source and the channel.

12. The flow cytometer of claim 1, further comprising at least one beam collector and at least one cone, each of the at least one beam collector and the at least one cone configured to receive at least some of at least one exit beam and at least one scatter beam from the at least one optical beam.

13. The flow cytometer of claim 1 further comprising at least one beam dump.

14. A flow cytometer, comprising:

a light source configured to emit at least one light beam along a first axis;

a channel configured to allow a particle-laden liquid to pass therethrough, the channel extending along a second axis; and

at least one detector configured to observe at least a point where the light beam and the liquid intersect,

wherein no masking or scattering bars are used such that the entire aperture of the at least one detector is exposed to the at least one light beam.

15. The flow cytometer of claim 14 wherein the second axis extends at an angle greater than 22 degrees and less than 70 degrees relative to the first axis.

16. The flow cytometer of claim 15 wherein the at least one detector comprises two spaced apart detectors.

17. The flow cytometer of claim 16 wherein the angle is between 34 ° -70 °.

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